NMR Structural Analysis of α-Bungarotoxin and Its Complex with the Principal α-Neurotoxin-binding Sequence on the α7 Subunit of a Neuronal Nicotinic Acetylcholine Receptor

Abstract

We report a new, higher resolution NMR structure of ␣-bungarotoxin that defines the structure-determining disulfide core and ␤-sheet regions. We further report the NMR structure of the stoichiometric complex formed between ␣-bungarotoxin and a recombinantly expressed 19-mer peptide ( The nicotinic acetylcholine receptor (nAChR)1 (1) is a ligandgated ion channel that mediates excitatory transmission at the neuromuscular junction and at synapses in the central and peripheral nervous systems. It is the most intensely studied member of the ligand-gated ion channel superfamily and serves as a model for understanding the structure and function of related ion conducting channels including glycine, ␥-aminobutyric acid type A, ␥-aminobutyric acid type C, and type 3 serotonin receptors. nAChRs are pentameric complexes that assemble in the membrane with 5-fold symmetry. Each subunit contains an N-terminal extracellular domain about 200 amino acids long followed by four membrane-spanning segments (M1-M4) with an intracellular loop of variable length between M3 and M4. The second transmembrane region from each subunit contributes to the formation of the wall lining the channel pore. In muscle and Torpedo electric organ, the subunit composition is (␣1) 2 ␤␥␦ and (␣1) 2 ␤⑀␦ in embryonic and adult tissue, respectively (for review, see Ref.

A Novel Pharmatope Tag Inserted into the β4 Subunit Confers Allosteric Modulation to Neuronal Nicotinic Receptors

“…In either case, the functional blockade of ␤4/␣1 [11]Loop-C subunit-containing nAChRs by Bgtx supports the view that rigid body-like subunit movements at subunit interfaces play an important role in receptor gating. Whether such movements (4), and the position of bound Bgtx is based on NMR studies using a Bgtx-binding peptide fragment of the rat ␣7 homomeric receptor (8). The extracellular domain of the ␤4/ ␣1 [11]Loop-C subunit is shown on the left, contributing its (ϩ) face to the Bgtx binding site, where Loop C, with Bgtx in close proximity below, is shown extending toward the ␣3 subunit on the right.…”

“…We replaced 7 contiguous residues from the presumed turn 7 of Loop C in ␤4 (i.e. VNPQDPS) with a sequence that we expected, based on NMR studies of receptor peptide fragments, to be sufficient to generate a high affinity Bgtx binding site (8,13,14). Our candidate pharmatope sequence (WVYYTCCPDTP) was derived from the tip of Loop C in the Torpedo ␣1 subunit.…”

“…In heteromeric nicotinic receptors, the adjacent subunit is always a non-␣ (4 -6). There is considerable evidence that Bgtx binding to the Loop C region prevents the association of acetylcholine (ACh) to its binding site, also in close proximity to Loop C, and as a consequence, the conformational changes leading to channel gating are precluded (7)(8)(9)(10).…”

Inhibition Mechanism of the Acetylcholine Receptor by α-Neurotoxins as Revealed by Normal-Mode Dynamics

“…It binds to the muscle AChR competitively, inhibiting ACh binding, thereby preventing channel opening and blocking neuromuscular transmission. The binding mode of α-neurotoxins to the AChR has been studied extensively (12)(13)(14)(15). In these structures, the AChR peptide segment α 1 W184-α1 D200 2 forms multiple interactions with finger I ( B T6-B S12) and II ( B M27-B L42) as well as the C terminus ( B H68-B R72) of αBTX.…”

“…In these structures, the AChR peptide segment α 1 W184-α1 D200 2 forms multiple interactions with finger I ( B T6-B S12) and II ( B M27-B L42) as well as the C terminus ( B H68-B R72) of αBTX. Several groups have attempted to construct a model of α-neurotoxins interacting with the extracellular domain of AChR (12,13,(15)(16)(17)(18) based on the X-ray structure of AChBP (25) and the cryo-EM structure of AChR (19). Our approach, was to use the structure of an AChR peptide in complex with αBTX and the structure of AChBP to construct a model of the AChR in complex with αBTX (15).…”